One of the most intriguing new paradigms in cancer biology is that of the cancer stem cell (CSC). The underlying hypothesis is that tumors contain a small population of cells with perpetual self-renewal capacity and that these cells initiate and maintain neoplastic growth. In addition, similarities between phenotypes and expression profiles support that some CSC's may actually be derived from the endogenous tissue stem cells. Based on this premise, this research proposal aims to address the hypothesis that Myc-induced liver tumors correspond with transformation of an endogenous stem cell that gives rise to CSC's. For the proposed research, a mouse model of Myc-induced liver cancer will be utilized to identify the associated CSC population that gives rise to the tumor. The first goal will be to identify the stem cells in normal liver and hepatocellular carcinoma. Multiple antibodies that are believed to recognize the various cells of the liver, including the endogenous tissue stem cell, will be tested for their ability to identify these populations with subsequent flow cytometry to isolate these cells from normal and tumor tissue. The second aim of this proposal will be to assess the self-renewal capacity of the putative liver tissue and cancer stem cells. Subcutaneous injection of the cells isolated in the previous aim into immuno-deficient mice will allow for determination of the tumorigenic potential of the separate populations. Similarly, transplantation into mice following liver damage will allow for the assessment of the ability of the cells to repopulate this organ. In order to monitor cell growth and localization, luciferase expressing cells will be used for transplantions with subsequent bioluminescent imaging: Finally, using the doxycycline-responsive, inducible system, the role of the Myc oncogene in the expansion and differentiation of normal and tumorigenic stem cells will be tested both in vivo and in vitro. These experiments include comparing the proliferation and tumorigenicity of these cells with activated or inactivated Myc. Additionally, insight into collaborating signaling pathways a will be investigated using multi-color, intracellular flow cytometry analysis. Together, it is believed that the proposed experiments will result in the isolation and characterization of the liver cancer stem cell. The ultimate consequences of this study would be to gain insight into the development of liver cancer, while potentially identifying novel therapeutic targets for this especially lethal disease.